The proposed research focuses on both reversible and irreversible mitochondrial inner membrane functional changes which occur in ischemic and autolyzing myocardium. The first aim of our planned work is the further characterization of the reversible inhibition of the mitochondrial ATPase which occurs in ischemic myocardium apparently as a direct result of lowered cell pH in the absence of electron flow. The second aim is the determination of the mechanism of the reactivation of the inhibited ATPase upon reperfusion in situ or upon state 4 re-energization in vitro. The third aim is the determination of the time course of the loss of mitochondrial competence, i.e., their ability to generate a transmembrane electrochemical gradient. The last aim is the determination of the relative and combined contributions of tissue acidosis and of tissue ATP depletion to the onset and progression of irreversible mitochondrial functional impairment which occurs during myocardial autolysis. Our data thus far suggest the following hypothesis, that the underlying mechanism of the ATPase inhibition is the reversible association of the ATPase inhibitor protein (AIP) of Pullman and Monroy which acts to block a dissipative ATP hydrolysis by the undriven ATP sysnthetase (in ischemia) but does not prevent renewed ATP synthesis upon re-energization (reflow). Other data suggest that beyond 20 min of myocardial autolysis, an increasing, irreversible impairment of electron flow at the NADH-CoQ reductase site may prevent total ATPase reactivation upon reflow or attempted re-energization. The ATPase inhibition work will be approached using submitochondrial particle (SMP) preparations of the following types: (1) control, (2) autolyzed, (3) energized, and (4) AIP-depleted. Immunoradiometric detection of the AIP on SMP will be used in conjunction with ATPase activity and kinetics measurements to verify the relationship between ATPase activity regulation and the presence or affinity of the AIP in the four types of SMP preparations to be used: The ability of ischemically damaged mitochondria to generate a membrane potential will be determined using their ability to reactivate their inhibited ATPase upon re-energization, as well as by an independent spectrophotometric method utilizing the dye, safronin O. The characterization of the reversible, regulatory inhibition of the mitochondrial ATPase is potentially of central importance to our understanding of the ability of heart muscle to react to oxygen depletion and acidosis.